Dihydrodipicolinate reductase (DHPR) is an enzyme constituent of the diaminopimelate-lysine pathway in bacteria and plants. It is a 273 residue polypeptide, with a molecular weight of about 28,000 Da, and has been shown to exist as a tetramer of identical subunits. The enzyme catalyzes the NADPH or NADH-dependent reduction of dihydrodipicolinate: ##STR1## Diaminopimelic acid (DAP) is an essential component of the peptidoglycan layer of bacterial cell walls, and disruption of its biosynthesis results in cell death, most probably due to instability of the peptidoglycan. Nine enzymes, including dihydrodipicolinate reductase, are involved in the biosynthesis of DAP. The presence of these enzymes is essential for the survival of bacteria and plants. These enzymes are absent in mammalian cells.
There are three known pathways for the biosynthesis of DAP utilized by bacteria (see FIG. 1). They have been designated as the succinylase, dehydrogenase and acetylase pathways. Different bacteria utilize one or more of the three pathways. For example, Escherichia coli synthesizes DAP predominantly via the succinylase pathway; Bacillus species show a great deal of variability in DAP biosynthesis, and the dehydrogenase and acetylase pathways appear to be the most common; and Corynebacterium species appear to utilize all three of the pathways. This redundancy may indicate the importance of the DAP biosynthesis for bacterial survival.
In the last few years, an increasing number of bacteria have developed drug resistance to many commonly used antibiotics, such as streptomycin, .beta.-lactams, isoniazid and ethionamide, thereby contributing to a new spread of disease. The search for new antibacterial drugs has consequently increased in importance. The nine enzymes of the DAP biosynthetic pathway represent excellent targets for the development of novel anti-bacterial and herbicidal agents, since no presently used antibiotics or herbicides target enzymes in this pathway. Inactivation of any of the enzymes would result in cell death. Inactivation of dihydrodipicolinate reductase will eliminate viability of bacteria and plants, since its product (THP) is a precursor of all of the three pathways.
It is desirable to know the three dimensional structure of dihydrodipicolinate reductase in order to develop compounds (drugs or herbicides) which inhibit the biochemical activity of this enzyme. If dihydrodipicolinate reductase crystals suitable for x-ray diffraction studies are produced, then the three dimensional structure of dihydrodipicolinate reductase can be determined. Obtaining the three dimensional structure of dihydrodipicolinate reductase would enable understanding the interactions between the enzyme (dihydrodipicolinate reductase) and substrates, which would enable those skilled in the art to utilize rational mechanism-based and structure-based drug design technology to develop specific inhibitors that would function as novel antibiotic drugs and herbicides.
It is therefore an object of this invention to provide dihydrodipicolinate reductase crystals which can be used to determine the three dimensional structure of dihydrodipicolinate reductase.
It is another object of this invention to provide methods of producing dihydrodipicolinate reductase crystals.
It is a further object of this invention to provide isolated dihydrodipicolinate reductase.
It is a still further object of this invention to provide a method of determining the three dimensional structure of dihydrodipicolinate reductase.
It is another object of this invention to provide the three dimensional structure of dihydrodipicolinate reductase, which structure can be used to develop bacteriocides and herbicides.